Angiotensin receptor autoantibodies as exposures that modify disease progression: Cross sectional, longitudinal and in vitro studies of prostate cancer.

Circulating angiotensin type I receptor (AT1R) agonistic autoantibodies (AT1RaAbs) that bind and chronically activate the receptor have been associated with a number of diseases suggesting that while the autoantibodies are not necessarily causative they may promote disease progression. The prostate has a local renin angiotensin system. The current study examines associations between AT1RaAbs and prostate cancer (PCA), disease-free survival (DFS), overall survival (OS) and AT1RaAb effects on PCA cell phenotype. In a cross-sectional set of serum obtained from 151 men diagnosed with PCA, nonmalignant prostate disease or no disease, higher serum AT1RaAb levels were associated with PCA and non-organ confined PCA. The odds ratio for PCA was 6.3 (95% confidence interval 2.2 to 18) for a positive 1:1600 titer and 18 (95% confidence interval 6.9 to 45) at AT1RaAb levels > 1.04 µg/ml, (p < 0.0001). In a longitudinal set of pre-diagnosis samples from 109 men, DFS hazard ratios of 2.2 (95% confidence interval 1.4 to 3.5) and 1.6 (95% confidence interval 1.0 to 2.5) for most proximal to diagnosis and most distal to diagnosis samples, respectively, were found for high versus low AT1RaAb groups. Hazard ratios for OS in most proximal and distal samples were 2.4 (95% confidence interval 1.6 to 3.6) and 1.8 (95% confidence interval 1.1 to 2.8), respectively. Accelerated failure modeling of survival indicated that a 1 µg/ml increase in AT1RaAb levels was associated with a reduction of DFS and OS by 20% at the most proximal time point and by 15% at the most distal time points. Adjusting for age, did not affect the association with DFS in proximal samples but changed distal time point DFS and OS to a 10% decrease for every 1 µg/ml increase in AT1RaAb. Additional adjustments for body mass index, systolic blood pressure and prostate-specific antigen did not appreciably alter these associations. AT1RaAb treatment of PC3, DU145, and LNCaP cells significantly increased the maximal growth rate approximately 2-fold and invasiveness approximately 3-fold. Conclusions: These observations provide evidence supporting AT1RaAbs as exposures that may modify prostate cancer progression and indicate they may be predictive markers for risk stratification.

Chitotriosidase, a marker of innate immunity, is elevated in patients with primary breast cancer.

BACKGROUND: Cancer progression has been associated with altered immune cell function and activation. Neopterin, which is secreted by interferon-γ stimulated macrophages, exhibits an association with multiple cancer types and metastatic disease. Chitotriosidase, which is secreted by chronically activated macrophages and granulocyte-macrophage colony-stimulating factor stimulated neutrophils has not been studied in the setting of cancer. OBJECTIVE: The goal of this discovery study was to screen chitotriosidase for diagnostic capacity in detecting cancer and compare its operating characteristics with those of neopterin. METHODS: Serum from subjects with breast (n= 66) or prostate (n= 70) cancer, and from 204 subjects free of malignant disease were studied. Chitotriosidase was measured by enzyme activity assay, while neopterin was measured by a competitive enzyme immunoassay. Statistical analyses included group comparisons by Mann Whitney U test, diagnostic capacity by receiver operating characteristics (ROC) curve analysis and biomarker associations with physiologic and clinical measures by Spearman correlation. RESULTS: Chitotriosidase activity was significantly higher in both cancer types compared with gender matched controls, though only in breast cancer was the diagnostic capacity significant (area under the ROC curve of 0.97 ± 0.01). In contrast, neopterin was significantly elevated in prostate cancer and exhibited discriminatory capacity (area under the ROC curve of 0.76 ± 0.05). Age, BMI, % body fat and metastasis were variables that correlated with neopterin, but not chitotriosidase levels. CONCLUSIONS: The operating characteristics of serum chitotriosidase were different from neopterin and further analysis of chitotriosidase as a biomarker for breast cancer is warranted.

Discovery and Validation of Agonistic Angiotensin Receptor Autoantibodies as Biomarkers of Adverse Outcomes.

BACKGROUND: Agonistic angiotensin II type 1 receptor autoantibodies (AT1RaAbs) have not been associated with functional measures or risk for adverse health outcomes. AT1RaAbs could be used to stratify patient risk and to identify patients who can benefit from angiotensin receptor blocker treatment. METHODS: Demographic and physiological covariates were measured in a discovery set of community-dwelling adults from Baltimore (N=255) and AT1RaAb associations with physical function tests and outcomes assessed. A group from Chicago (N=60) was used for validation of associations and to explore the impact of angiotensin receptor blocker treatment. RESULTS: The Baltimore group had 28 subjects with falls, 32 frail subjects, and 5 deaths. Higher AT1RaAbs correlated significantly with interleukin-6 (Spearman r=0.33, P<0.0001), systolic blood pressure (Spearman r=0.28, P<0.0001), body mass index (Spearman r=0.28, P<0.0001), weaker grip strength (Spearman r=-0.34, P<0.01), and slower walking speed (Spearman r=-0.30, P<0.05). Individuals with high AT1RaAbs were 3.9 (95% confidence interval, 1.38-11.0) times more likely to be at high risk after adjusting for age (P<0.05). Every 1 µg/mL increase in AT1RaAbs increased the odds of falling 30% after adjusting for age, sex, body mass index, and blood pressure. The Chicago group had 46 subjects with falls and 60 deaths. Serum AT1RaAb levels were significantly correlated with grip strength (Spearman r=-0.57, P<0.005), walking speed (Spearman r=-0.47, P<0.005), and falls (Spearman r=0.30, P<0.05). Every 1 µg/mL increase in AT1RaAbs, decreased time to death by 9% after adjusting for age, sex, body mass index, and blood pressure. Chronic treatment with angiotensin receptor blockers was associated with better control of systolic blood pressure and attenuation of decline in both grip strength and time to death. CONCLUSIONS: In older individuals, higher AT1RaAb levels were associated with inflammation, hypertension, and adverse outcomes. Angiotensin receptor blocker treatment may blunt the harm associated with high levels of AT1RaAb.

Survey of the use of domperidone and the association with QTc prolongation in general practice.

PURPOSE OF THE STUDY: Domperidone is associated with QTc prolongation, predisposing to the development of ventricular arrhythmias. In 2014, The Medicines and Healthcare Regulatory Agency (MHRA) recommended restricting its use. We assessed whether these recommendations have been implemented in a general practice. STUDY DESIGN: We conducted a prospective study using the general practitioner (GP) computer database on patients who had at least one repeat prescription for domperidone in 12 months. Data were presented to the doctors and the survey was repeated 7 months later. RESULTS: Sixty-four patients (mean age 61.3±16.4 years) were identified who had received at least one repeat prescription of domperidone. Twenty patients were being prescribed over the recommended daily dose. Nineteen patients were coprescribed medications known to prolong the QTc interval and two CYP3A4 inhibitors. The repeat survey performed 7 months later demonstrated a 70% reduction in the number of patients prescribed domperidone to a total of 19 (three patients prescribed above the recommended dose) none of which had a history of cardiac disease or were being coprescribed drugs known to prolong the QTc interval. CONCLUSIONS: Following the publication of the MHRA recommendations and presentation of our initial survey, there has been a significant reduction in the number of patients treated with domperidone and those coprescribed drugs known to prolong the QTc interval. We suggest that regular review of GP practice database should be performed to identify those patients prescribed domperidone and at risk of life-threatening arrhythmias and measures taken to use alternative pharmacological agents.

WISP1/CCN4: a potential target for inhibiting prostate cancer growth and spread to bone.

Prostate cancer (PC) is a leading cause of death in men however the factors that regulate its progression and eventual metastasis to bone remain unclear. Here we show that WISP1/CCN4 expression in prostate cancer tissues was up-regulated in early stages of the disease and, further, that it correlated with increased circulating levels of WISP1 in the sera of patients at early stages of the disease. WISP1 was also elevated in the mouse prostate cancer model TRAMP in the hypoplastic diseased tissue that develops prior to advanced carcinoma formation. When the ability of anti-WISP1 antibodies to reduce the spread of PC3-Luc cells to distant sites was tested it showed that twice weekly injections of anti-WISP1 antibodies reduced the number and overall size of distant tumors developed after intracardiac (IC) injection of PC3-Luc cells in mice. The ability of antibodies against WISP1 to inhibit growth of PC3-Luc cancer cells in mice was also evaluated and showed that twice weekly injections of anti-WISP1 antibodies reduced local tumor growth when examined in xenografts. To better understand the mechanism of action, the migration of PC3-Luc cells through membranes with or without a Matrigel™ barrier showed the cells were attracted to WISP1, and that this attraction was inhibited by treatment with anti-WISP1 antibodies. We also show the expression of WISP1 at the bone-tumor interface and in the stroma of early grade cancers suggested WISP1 expression is well placed to play roles in both fostering growth of the cancer and its spread to bone. In summary, the up-regulation of WISP1 in the early stages of cancer development coupled with its ability to inhibit spread and growth of prostate cancer cells makes it both a potential target and an accessible diagnostic marker for prostate cancer.

Serum chitotriosidase, a putative marker of chronically activated macrophages, increases with normal aging.

BACKGROUND: Chitotriosidase (ChT) is secreted by chronically activated macrophages in Gaucher's disease. We hypothesize that circulating levels of ChT are altered with normal aging, reflecting age-related chronic macrophage activation. Potential sources that might contribute to altered levels were assessed by measuring systemic levels of ChT are α-naphthyl acetate esterase, a macrophage lysosomal enzyme; granulocyte-macrophage colony-stimulating factor (GM-CSF), which stimulates neutrophilic granule release of ChT; interleukin-6 (IL-6); and neopterin, a macrophage activation marker. METHODS: Serum was obtained from 315 healthy participants whose age ranged from 18 to 92 years. Anthropometric measures included percent body fat and body mass index. ChT and α-naphthyl acetate esterase levels were measured by enzyme activity assays. GM-CSF, IL-6, and neopterin concentrations were measured by commercial enzyme-linked immunosorbent assays. Serum marker values were statistically analyzed using nonparametric tests. RESULTS: Six percent of the participants had undetectable ChT levels. A positive association with age was observed for ChT and IL-6, whereas a negative correlation with age was seen for α-naphthyl acetate esterase and GM-CSF. ChT values were not associated with α-naphthyl acetate esterase or GM-CSF levels. ChT was independently associated with IL-6 and neopterin levels, but statistical significance was attenuated when controlled for age. CONCLUSIONS: The data are consistent with increased serum ChT activity not arising from altered macrophage lysosomal enzyme trafficking or GM-CSF-stimulated release of neutrophil granule stores. The association of ChT with age remains significant after controlling for neopterin and IL-6 changes with age, suggesting that ChT levels reflect a macrophage state distinct from acute macrophage activation or inflammatory state.

Plasma neopterin level as a marker of peripheral immune activation in amnestic mild cognitive impairment and Alzheimer's disease.

OBJECTIVE: Alterations of the immune system play important roles in Alzheimer's disease (AD). The primary purpose of this study was to compare the plasma levels of neopterin, a marker of cellular immune activity, in amnestic mild cognitive impairment (aMCI), early (mild to moderate) AD, and cognitively normal controls. In addition, the correlation of plasma neopterin with interferon-gamma (IFN-γ) and interleukin-6 (IL-6) was also examined. METHODS: Plasma samples from patients with mild-to-moderate AD (N = 34), aMCI (N = 27), and cognitively normal controls (N = 30) were obtained from the Johns Hopkins Alzheimer's Disease Research Center. Plasma neopterin, IFN-γ, and IL-6 levels were measured using commercially available ELISAs. Multiple linear regression was performed to study differences in the baseline neopterin levels between normal, aMCI, and AD patients. Pearson correlation coefficients were estimated for neopterin and IFN-γ and IL-6 levels. All analyses were conducted using SAS (SAS Institute, Inc., Cary, NC) and GraphPad Prism version 5.00 for Window (GraphPad Software, San Diego, CA, USA). RESULTS: AD subjects had significantly higher neopterin values compared with aMCI (ß = 0.202, p = 0.004) and normal (ß = 0.263, p = 0.0004) subjects. There was no statistically significant difference between normal and aMCI subjects. Significant associations between neopterin and IFN-γ (r = 0.41, p < 0.0001) and IL-6 (r = 0.35, p = 0.0006) levels were found. CONCLUSIONS: Our study demonstrates that peripheral immune response may be stronger in later stages of AD pathophysiology, when dementia has developed.

Identification and characterization of a functional mitochondrial angiotensin system.

The renin-angiotensin (Ang) system regulates multiple physiological functions through Ang II type 1 and type 2 receptors. Prior studies suggest an intracellular pool of Ang II that may be released in an autocrine manner upon stretch to activate surface membrane Ang receptors. Alternatively, an intracellular renin-Ang system has been proposed, with a primary focus on nuclear Ang receptors. A mitochondrial Ang system has not been previously described. Here we report that functional Ang II type 2 receptors are present on mitochondrial inner membranes and are colocalized with endogenous Ang. We demonstrate that activation of the mitochondrial Ang system is coupled to mitochondrial nitric oxide production and can modulate respiration. In addition, we present evidence of age-related changes in mitochondrial Ang receptor expression, i.e., increased mitochondrial Ang II type 1 receptor and decreased type 2 receptor density that is reversed by chronic treatment with the Ang II type 1 receptor blocker losartan. The presence of a functional Ang system in human mitochondria provides a foundation for understanding the interaction between mitochondria and chronic disease states and reveals potential therapeutic targets for optimizing mitochondrial function and decreasing chronic disease burden with aging.

IL-6-independent association of elevated serum neopterin levels with prevalent frailty in community-dwelling older adults.

BACKGROUND: neopterin is a monocyte/macrophage-derived immune activation marker and its levels increase with age. Frailty is an important clinical syndrome of old age. Previous studies have shown significant association between elevated interleukin-6 (IL-6) levels and frailty. The objective of this study was to evaluate IL-6-independent association of serum neopterin levels with prevalent frailty. METHODS: this is a cross-sectional study in community-dwelling older adults recruited from residential and retirement communities in Baltimore, MD, USA. Frailty was determined using validated screening criteria. Serum neopterin and IL-6 levels were measured using standard enzyme-linked immunosorbent assay. Pearson correlation and multivariate linear regression analysis was performed to assess the relationship between log(neopterin) and log(IL-6). Odds ratios (ORs) for frailty were calculated using log(neopterin) and log(IL-6) as continuous measures and across tertiles of neopterin and IL-6 levels, adjusting for age, race, sex, education and body mass index. RESULTS: one hundred and thirty-three individuals with a mean age of 84 years (range 72-97) completed the study. Neopterin levels were significantly higher in frail older adults than those in non-frail controls [median: 8.94 versus 8.35 nM, respectively, P < 0.001 t-test on log(neopterin)]. Log(neopterin) was significantly associated with prevalent frailty, adjusting for log(IL-6). Participants in the top tertile of neopterin had OR of 3.80 [95% confidence interval (CI) = 1.36-10.6, P < 0.01] for frailty. As expected, participants in the top tertile of IL-6 had OR of 3.29 (95% CI = 1.21-7.86, P < 0.05) for frailty. Log(neopterin) correlated with log(IL-6) (correlation coefficient = 0.19, P < 0.05). Moreover, OR for participants in the top neopterin tertile remained significant after adjusting for IL-6 (OR = 3.97, 95% CI = 1.15-13.72, P < 0.05). CONCLUSION: elevated neopterin levels had IL-6-independent association with prevalent frailty, suggesting potential monocyte/macrophage-mediated immune activation in the frail elderly.

Comparison of conventional ELISA with electrochemiluminescence technology for detection of amyloid-ß in plasma.

Plasma amyloid-ß (Aß) level could be useful as a non-invasive biomarker in Alzheimer's disease research. We compared a multiplex electrochemiluminescence detection method with a well established ELISA method for plasma Aß quantification. Compared to the ELISA method, the electrochemiluminescence detection method demonstrates a statistically significant, but modest correlation. The reasons for this may include the differences in the affinities of antibodies, and purity and source of Aß peptides used as standards. However, the advantages of electrochemiluminescence detection technology include short processing time and small sample volume. This comparison demonstrates the need for a further study in optimizing this system.

Serum levels of the immune activation marker neopterin change with age and gender and are modified by race, BMI, and percentage of body fat.

BACKGROUND: Neopterin, a GTP metabolite expressed by macrophages, is a marker of immune activation. We hypothesize that levels of this serum marker alter with donor age, reflecting increased chronic immune activation in normal aging. In addition to age, we assessed gender, race, body mass index (BMI), and percentage of body fat (%fat) as potential covariates. METHODS: Serum was obtained from 426 healthy participants whose age ranged from 18 to 87 years. Anthropometric measures included %fat and BMI. Neopterin concentrations were measured by competitive ELISA. The paired associations between neopterin and age, BMI, or %fat were analyzed by Spearman's correlation or by linear regression of log-transformed neopterin, whereas overall associations were modeled by multiple regression of log-transformed neopterin as a function of age, gender, race, BMI, %fat, and interaction terms. RESULTS: Across all participants, neopterin exhibited a positive association with age, BMI, and %fat. Multiple regression modeling of neopterin in women and men as a function of age, BMI, and race revealed that each covariate contributed significantly to neopterin values and that optimal modeling required an interaction term between race and BMI. The covariate %fat was highly correlated with BMI and could be substituted for BMI to yield similar regression coefficients. CONCLUSION: The association of age and gender with neopterin levels and their modification by race, BMI, or %fat reflect the biology underlying chronic immune activation and perhaps gender differences in disease incidence, morbidity, and mortality.

Small integrin-binding proteins as serum markers for prostate cancer detection.

PURPOSE: The small integrin-binding ligand N-linked glycoprotein (SIBLING) gene family includes bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE), and osteopontin (OPN). Previous studies have separately reported elevated expression of BSP, OPN, or DSPP in prostate tumor paraffin sections. We hypothesized that SIBLINGs may be informative serum markers for subjects with prostate cancer. METHODS: Expression levels of SIBLINGs in biopsies of normal tissue and tumors from prostate were determined by cDNA array and by immunohistochemical staining with monoclonal antibodies. Competitive ELISAs for measuring total BSP, DSPP, MEPE, and OPN were applied to a test group of 102 subjects with prostate cancer and 110 normal subjects and a validation group of 90 subjects. RESULTS: BSP, DMP1, DSPP, and OPN exhibited elevated mRNA expression and protein levels in biopsies. BSP, DSPP, and OPN were elevated in serum from prostate cancer subjects, with serum DSPP exhibiting the greatest difference, yielding an area under the receiver operator characteristic curve value of 0.98. Serum BSP and OPN levels were significantly elevated only in late stages, whereas DSPP was significantly elevated at all stages. Optimal serum value cutoff points derived for BSP, OPN, and DSPP were applied as a validation test to a new group of 90 subjects and DSPP yielded a sensitivity of 90% and a specificity of 100%. CONCLUSION: Of the SIBLING gene family members, DSPP appears to be a strong candidate for use in serum assays for prostate cancer detection.

Serum markers of apoptosis decrease with age and cancer stage.

The physical manifestations of aging reflect a loss of homeostasis that effects molecular, cellular and organ system functional capacity. As a sentinel homeostatic pathway, changes in apoptosis can have pathophysiological consequences in both aging and disease. To assess baseline global apoptosis balance, sera from 204 clinically normal subjects had levels of sFas (inhibitor of apoptosis), sFasL (stimulator of apoptosis), and total cytochrome c (released from cells during apoptosis) measured. Serum levels of sFas were significantly higher while sFasL and cytochrome c levels were lower in men compared to women. With increasing age there was a decrease in apoptotic markers (cytochrome c) and pro-apoptotic factors (sFasL) and an increase in anti-apoptotic factors (sFas) in circulation. The observed gender differences are consistent with the known differences between genders in mortality and morbidity. In a separate cohort, subjects with either breast (n = 66) or prostate cancer (n = 38) exhibited significantly elevated sFas with reduced sFasL and total cytochrome c regardless of age. These markers correlated with disease severity consistent with tumor subversion of apoptosis. The shift toward less global apoptosis with increasing age in normal subjects is consistent with increased incidence of diseases whose pathophysiology involves apoptosis dysregulation.

Structural requirements for bone sialoprotein binding and modulation of matrix metalloproteinase-2.

Bone sialoprotein (BSP) has been shown to induce limited gelatinase activity in latent matrix metalloproteinase-2 (MMP-2) without removal of the propeptide and to restore enzymatic activity to MMP-2 previously inhibited by tissue inhibitor of matrix metalloproteinase-2 (TIMP2). The current study identifies structural domains in human BSP and MMP-2 that contribute to these interactions. The 26 amino acid domain encoded by exon 4 of BSP is shown by a series of binding and activity assays to be involved in the displacement of MMP-2's propeptide from the active site and thereby inducing the protease activity. Binding assays in conjunction with enzyme activity assays demonstrate that both amino- and carboxy-terminal domains of BSP contribute to restoration of activity to TIMP2-inhibited MMP-2, while the MMP-2 hemopexin domain is not required for reactivation.

Bone sialoprotein binding to matrix metalloproteinase-2 alters enzyme inhibition kinetics.

Bone sialoprotein (BSP) is a secreted glycophosphoprotein normally restricted in expression to skeletal tissue that is also induced by multiple neoplasms in vivo. Previous work has shown that BSP can bind to matrix metalloproteinase-2 (MMP-2). Because of MMP-2 activity in promoting tumor progression, potential therapeutic inhibitors were developed, but clinical trials have been disappointing. The effect of BSP on MMP-2 modulation by inhibitors was determined with purified components and in cell culture. Enzyme inhibition kinetics were studied using a low-molecular weight freely diffusable substrate and purified MMP-2, BSP, and natural (tissue inhibitor of matrix metalloproteinase-2) and synthetic (ilomastat and oleoyl- N-hydroxylamide) inhibitors. We determined parameters of enzyme kinetics by varying substrate concentrations at different fixed inhibitor concentrations added to MMP-2 alone, MMP-2 and BSP, or preformed MMP-2-BSP complexes and solving a general linear mixed inhibition rate equation with a global curve fitting program. Two in vitro angiogenesis model systems employing human umbilical vein endothelial cells (HUVECs) were used to follow BSP modulation of MMP-2 inhibition and tubule formation. The presence of BSP increased the competitive K I values between 15- and 47-fold for natural and synthetic inhibitors. The extent of tubule formation by HUVECs cocultured with dermal fibroblasts was reduced in the presence of inhibitors, while the addition of BSP restored vessel formation. A second HUVEC culture system demonstrated that tubule formation by cells expressing BSP could be inhibited by an activity blocking antibody against MMP-2. BSP modulation of MMP-2 activity and inhibition may define its biological role in promoting tumor progression.

Elevated levels of neopterin in sleep-disordered breathing.

BACKGROUND: Sleep-disordered breathing (SDB) is increasingly being recognized as an independent risk factor for hypertension and cardiovascular disease. Recent evidence suggests that the maladaptive physiologic response to SDB, particularly cardiovascular effects, may result in part from systemic inflammation. Although abnormal cytokine levels have been documented in SDB, data on whether SDB is associated with cellular activation are limited. Thus, this investigation sought to determine whether neopterin, a marker released by activated macrophages, is increased in SDB. METHODS AND RESULTS: Fifty-five men, free of medical comorbidity, undergoing polysomnography had fasting serum tested for neopterin levels. Multivariable regression methods were used to quantify the association between neopterin and quartiles of the apnea hypopnea index (AHI) while accounting for body mass index, waist circumference, and percentage of body fat. Quartiles of AHI (I: < 3.83 events per hour; II: 3.83 to 11.98 events per hour; III: 11.99 to 36.82 events per hour; IV > 36.82 events per hour) indicated a range from no SDB through severe SDB. Compared to the subjects in the first AHI quartile, serum neopterin levels were higher by 3.0%, 10.9%, and 26.5% in the second, third, and fourth AHI quartiles, respectively (p < 0.001for linear trend). Neopterin levels also were higher in those with greater degree of sleep-related hypoxemia, more stage 1 sleep, and less stage 2 sleep. CONCLUSION: The results of this study indicate that severity of SDB independently associates with serum levels of neopterin, a marker for macrophage activation that may play an important role in the pathogenesis of SDB-related cardiovascular disease.

The role of mutant UDP-N-acetyl-alpha-D-galactosamine-polypeptide N-acetylgalactosaminyltransferase 3 in regulating serum intact fibroblast growth factor 23 and matrix extracellular phosphoglycoprotein in heritable tumoral calcinosis.

CONTEXT: Familial tumoral calcinosis (TC) results from disruptions in phosphate metabolism and is characterized by high serum phosphate with normal or elevated 1,25 dihydroxyvitamin vitamin D concentrations and ectopic and vascular calcifications. Recessive loss-of-function mutations in UDP-N-acetyl-alpha-D-galactosamine-polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) and fibroblast growth factor-23 (FGF23) result in TC. OBJECTIVE: The objective of the study was to determine the relationship between GALNT3 and FGF23 in familial TC. DESIGN, SETTING, AND PATIENTS: We assessed the major biochemical defects and potential genes involved in patients with TC. INTERVENTION: Combination therapy consisted of the phosphate binder Sevelamer and the carbonic anhydrase inhibitor acetazolamide. RESULTS: We report a patient homozygous for a GALNT3 exon 1 deletion, which is predicted to truncate the encoded protein. This patient had high serum FGF23 concentrations when assessed with a C-terminal FGF23 ELISA but low-normal FGF23 levels when tested with an ELISA for intact FGF23 concentrations. Matrix extracellular phosphoglycoprotein has been identified as a possible regulator of phosphate homeostasis. Serum matrix extracellular phosphoglycoprotein levels, however, were normal in the family with GALNT3-TC and a kindred with TC carrying the FGF23 S71G mutation. The tumoral masses of the patient with GALNT3-TC completely resolved after combination therapy. CONCLUSIONS: Our findings demonstrate that GALNT3 inactivation in patients with TC leads to inadequate production of biologically active FGF23 as the most likely cause of the hyperphosphatemic phenotype. Furthermore, combination therapy may be effective for reducing the tumoral burden associated with familial TC.

Bone sialoprotein mediates the tumor cell-targeted prometastatic activity of transforming growth factor beta in a mouse model of breast cancer.

Transforming growth factor betas (TGF-beta) play a dual role in carcinogenesis, functioning as tumor suppressors early in the process, and then switching to act as prometastatic factors in late-stage disease. We have previously shown that high molecular weight TGF-beta antagonists can suppress metastasis without the predicted toxicities. To address the underlying mechanisms, we have used the 4T1 syngeneic mouse model of metastatic breast cancer. Treatment of mice with a monoclonal anti-TGF-beta antibody (1D11) significantly suppressed metastasis of 4T1 cells to the lungs. When metastatic 4T1 cells were recovered from lungs of 1D11-treated and control mice, the most differentially expressed gene was found to be bone sialoprotein (Bsp). Immunostaining confirmed the loss of Bsp protein in 1D11-treated lung metastases, and TGF-beta was shown to regulate and correlate with Bsp expression in vitro. Functionally, knockdown of Bsp in 4T1 cells reduced the ability of TGF-beta to induce local collagen degradation and invasion in vitro, and treatment with recombinant Bsp protected 4T1 cells from complement-mediated lysis. Finally, suppression of Bsp in 4T1 cells reduced metastasis in vivo. We conclude that Bsp is a plausible mediator of at least some of the tumor cell-targeted prometastatic activity of TGF-beta in this model and that Bsp expression in metastases can be successfully suppressed by systemic treatment with anti-TGF-beta antibodies.

Fibroblast growth factor-23 is regulated by 1alpha,25-dihydroxyvitamin D.

UNLABELLED: Serum FGF-23 regulation was studied in patients with hypoparathyroidism or pseudohypoparathyroidism treated with calcitriol. Serum FGF-23 levels changed in parallel in response to changes in serum 1,25-D, suggesting that FGF-23 may be regulated by 1,25-D. In addition, the phosphaturic effect of FGF-23 may be diminished in the absence of PTH action on the kidney. INTRODUCTION: Fibroblast growth factor (FGF)-23 is a recently described hormone that has been shown to be involved in the regulation of phosphate and vitamin D metabolism. The physiologic role of FGF-23 in mineral metabolism and how serum FGF-23 levels are regulated have yet to be elucidated. Three patients with mineral metabolism defects that allowed for the investigation of the regulation of FGF-23 were studied. MATERIALS AND METHODS: Patient 1 had postsurgical hypoparathyroidism and Munchausen's syndrome and consumed a pharmacologic dose of calcitriol. Patient 2 had postsurgical hypoparathyroidism and fibrous dysplasia of bone. She was treated with increasing doses of calcitriol followed by synthetic PTH(1-34). Patient 3 had pseudohypoparathyroidism type 1B and tertiary hyperparathyroidism. She underwent parathyroidectomy, which was followed by the development of hungry bone syndrome and hypocalcemia, requiring treatment with calcitriol. Serum FGF-23 and serum and urine levels of mineral metabolites were measured in all three patients. RESULTS: Patient 1 had an acute and marked increase in serum FGF-23 (70 to 670 RU/ml; normal range, 18-108 RU/ml) within 24 h in response to high-dose calcitriol administration. Patient 2 showed stepwise increases in serum FGF-23 from 117 to 824 RU/ml in response to increasing serum levels of 1alpha,25-dihydroxyvitamin D (1,25-D). Finally, before parathyroidectomy, while hypercalcemic, euphosphatemic, with low levels of 1,25-D (10 pg/ml; normal range, 22-67 pg/ml), and with very high serum PTH (863.7 pg/ml; normal range, 6.0-40.0 pg/ml), patient 3 had high serum FGF-23 levels (217 RU/ml). After surgery, while hypocalcemic, euphosphatemic, and with high serum levels of serum 1,25-D (140 pg/ml), FGF-23 levels were higher than preoperative levels (305 RU/ml). It seemed that the phosphaturic effect of FGF-23 was diminished in the absence of PTH or a PTH effect. CONCLUSIONS: Serum FGF-23 may be regulated by serum 1,25-D, and its phosphaturic effect may be less in the absence of PTH.